Identification, purification and detection of WSBV baculovirus associated with white spot syndrome

ABSTRACT

This invention relates to the identification, purification and detection of a new infectious viral agent in arthropods, especially shrimps. The virus is named as WSBV (Baculovirus associated with white spot syndrome). Two WSBV genomic DNA libraries were constructed and based upon the sequence of one of the cloned WSBV DNA fragments, a WSBV specific primer set for PCR to detect the WSBV infection in penaeid shrimps has been developed. The results of the present invention provide an effective diagnostic tool for screening WSBV infection in animal host organisms, in particular shrimps, to prevent the further spread of this viral disease.

This is a divisional of U.S. application Ser. No. 08/587,670, filed Jan.17, 1996, U.S. Pat. No. 5,824,535.

FIELD OF INVENTION

This invention relates to the identification, purification and detectionof a new infectious viral agent in arthropods, especially shrimps. Thevirus is named as WSBV (Baculovirus associated with white spotsyndrome).

BACKGROUND OF THE INVENTION

Recently, disease outbreaks have caused mass mortality among culturedpenaeid shrimps in Asian countries. Since 1992, outbreaks of a newdisease leading to serious mortality among populations of culturedkuruma shrimp (Penaeus japonicus) have occurred in northern Taiwan. Thedisease is characterized by obvious white spots on the carapace,appendages and the inside surface of the body, and cumulative mortalityreaches 100% within 2-7 days. The diseased shrimps also display signs oflethargy and reddish coloration of the hepatopancreas. In 1993, whitespot syndrome (W.S.S.) in cultured giant tiger prawn (P. monodon) andredtail prawn (P. penicillatus) was observed. Serious damage to penaeidshrimp production by W.S.S. in Taiwan has been reported (Tung et al.personal communication).

An epizootiological survey of kuruma shrimp in Japan reports similarfindings (Nakano et al., Fish Pathology, 29 (2):135-139, 1994).According to the evidence from electron microscopy and the results ofchallenge tests with the filtrate from diseased shrimp lymphoid organs,the causative agent was a virus that was temporarily designated RV-PJ, arod-shaped nuclear virus of Penaeus japonicus (Inouye et al., FishPathology, 29 (2):149-158, 1994; Takahashi et al., Fish Pathology, 29(2):121-125, 1994).

To date, the prevalence of baculoviruses in cultured penaeid shrimps hasbeen well documented (Lightner et al., Aquaculture, 32: 209-233, 1983).Among these penaeid baculoviruses, monodon baculovirus (MBV),baculoviral mid-gut necrosis virus (BMNV) and Baculovirus penaei (BP)were considered to be the most important because they have on occasionscaused serious losses in infected shrimp populations (Couch, Nature 247(5438): 22-231, 1974; J. Invertebr. Pathol., 24:311-331, 1974; Lightner& Redman, J. Invertebr. Pathology, 38: 299-302, 1981; Lightner et al.(1983), supra; Lightner et al., 1987; Sano et al., Fish Pathology, 15:185-191, 1981).

Baculovirus-like viral particles were observed in the spontaneouslydiseased penaeid shrimp with W.S.S. (Tung et al., personalcommunication). This virus may possibly be the main causative agent forthe W.S.S. that has occurred in Taiwan in penaeid shrimps. To preventthe spread of W.S.S. in shrimps, thereby rescuing the financial lossescaused by this viral disease, it is necessary to identify the actualcausative agent and then to develop a diagnostic method that is easy,accurate and not time-consuming in the detection of W.S.S. withoutsacrificing the whole subjects tested.

SUMMARY OF THE INVENTION

This invention is based on the finding of a new causative agentresponsible for the incidence of white spot syndrome in penaeid shrimps.The causative agent has been isolated and purified and found to be anon-occluded rod-shaped virus particle, which is enveloped, 330±20 nm inlength and 87±7 nm in diameter. This virus is determined to be a memberof genus NOB (Non-Occluded Baculovirus) of the subfamilyNudibaculovirinae of Baculoviridae and the present isolate is designatedas PMNOBIII, and as WSBV (Baculovirus associated with White Spotsyndrome) to indicate PmNOBIII related agents. A WSBV genomic DNAlibrary was constructed and based upon the sequence of one of the clonedWSBV DNA fragments, a WSBV specific primer set for PCR to detect theWSBV infection in penaeid shrimps has been developed. By PCR with theWSBV specific primer set, it was demonstrated that the causative agentsof white spot syndrome in different shrimp species are in fact closelyrelated. The results of the present invention provide an effectivediagnostic tool for screening WSBV infection in animal host organisms,in particular shrimps, which tool may be extremely important inpreventing the further spread of this viral disease. An easy, sensitiveand specific ready-to-use diagnostic product, which includes primersestablished based on the nucleotide sequence of a unique genomic DNAclone derived from WSBV, can be developed for the detection of thepresence of WSBV and to halt the further spread of this viral disease.The WSBV (also called PmNOBIII) described herein was deposited under theterms of the Budapest Treaty on Jan. 11, 1996, with the China Center forType Culture Collection, Wuhan University, Luo Jia Shan, Wuhan, Hubei,430072, People's Republic of China, where the deposit was givenaccession number CCTCC-V96001.

Features and advantages of the present invention will become apparent inthe following detailed description of the preferred embodiments, withreference to the accompanying drawings, of which:

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 is a photograph of Penaeus monodon with white spot syndromeshowing the white spots from barely visible to 3 mm in diameter. Bar: 1cm.

FIG. 2 is a light micrograph of cuticular epidermis under thecephalothorax exoskeleton (C) from Penaeus monodon with white spotsyndrome showing basophilic inclusions in hypertrophied nuclei ofdegenerated cells (arrows). Bar: 10 μm.

FIG. 3 is a transmission electron micrograph of thin-sectioned infectedtissues underneath the cephalothoracic exoskeletal cuticle (C) fromPenaeus monodon with white spot syndrome showing virus particles in thenecrotic area and in a hypertrophied nucleus (Arrow). Bar: 0.5. μm.

FIG. 4 shows rod-shaped viral particles in the epidermis ofexperimentally infected P. japonicus with the filtrate obtained fromdiseased P. monodon) Scale bar=200 nm.

FIG. 5 is a negatively stained micrograph of the pellet from filtrate ofdiseased P. monodon epidermis. Virus particles (arrow) with rod-shapemorphology were observed. Scale bar=500 nm.

FIG. 6 displays the white spot (arrow) on the removed carapace ofexperimentally infected P. japonicus.

FIG. 7 shows the cumulative mortalities (%) of P. japonicus (0.08 gaverage weight) experimentally infected by immersion in filtrates fromdiseased P. japonicus and P. monodon contrasted with healthy shrimpcontrol group(s).

FIG. 8 is a transmission electron micrograph of negatively stainedpurified virions showing a tail-like projection (P) extending from oneend of the virus. Bar: 0.1 μm.

FIG. 9 is a transmission electron micrograph of Negatively stainednon-enveloped nucleocapsid showing the cross-striations on the capsidformed by the ring subunits (arrows). The rings align perpendicularly tothe longitudinal axis of the capsid. Bar: 0.1 μm.

FIG. 10 shows an ethidium bromide-stained agarose gel of PmNOBIII DNAextracted from purified virions. A single molecule of DNA is observed inthe gel. Lane 1: lamda phage DNA HindIII fragment marker; lanes 2-4:extracted PmNOBIII DNA from each of three respective preparations.

FIG. 11 shows an ethidium bromide-stained agarose gel of PmNOBIII DNAdigested with three restriction endonucleases. At least twenty-two DNAfragments (arrows) can be identified in this gel. Lane 1: lamda phageDNA HindIII fragment marker; lane 2: PmNOBIII DNA HindIII fragments;lane 3: PmNOBIII DNA SalI fragments; lane 4: PMNOBIII DNA XhoIfragments; and lane 5: 1 Kb DNA Ladder.

FIG. 12 shows an ethidium bromide-stained agarose gel of PCR-amplified18S rDNA fragment from shrimp genomic DNA. Two primers for highlyconserved regions of the 18S rRNA sequence of decapods, 143F and 145R,were used for the reaction and primed the amplification of the 848-bpfragment from DNA template prepared from the healthy Penaeus monodon(lane 2). Lane 1, pGEM DNA size marker. The size of DNA markers isindicated in base pairs (bp).

FIG. 13 shows a qualitative assessment using PCR and shrimp DNA specificprimer set 143F and 145R, for monitoring shrimp DNA contamination in theWSBV genomic DNA preparations. The PCR products were analyzed on a 1%agarose gel. The shrimp DNA contamination is evidenced by the presenceof a 848 bp PCR product. Lane 1, pGEN DNA size marker ; lanes 2-6, WSBVgenomic DNA preparations as DNA template; lanes 7-8, shrimp genomic DNAprepared from healthy Penaeus monodon (lane 7) and P. japonicus (lane 8)as DNA template; lane 9: without DNA template. The size of the of DNAmarkers is given in base pairs (bp).

FIG. 14 shows the SalI digested WSBV DNA fragments. WSBV genomic DNA wasdigested with SalI restriction endonuclease at 37° C. for 3 hr. A 5-μlaliquot was analyzed on a 0.8% agarose gel containing ethidium bromideshowing the fragments with a size from 15 kbp to less than 1 kbp (lane2). From the same batch of digested DNA, a 20-μl aliquot was used forWSBV DNA library construction. Lane 1, lambda phage DNA HindIII fragmentmarker. The size of DNA markers is indicated in base pairs (bp).

FIG. 15A displays a diagram of the SalI-1461 bp DNA fragment cloned inplasmid pms146 and FIG. 15B shows the locations of the primers, whichare used for PCR amplication, in the SalI-1461 bp DNA fragment. The146F1 and 146R1 prime the amplification of a 1447-bp fragment, while146F2 and 146R2 prime the amplification of a 941-bp fragment. Thepositions of two EcoRI sites in SalI 1461 bp DNA fragment are alsoindicated. FIG. 15C shows the detailed nucleotide sequence of theSalI-1461 bp fragment, in which the locations of the two primer sets146F1/146R1 and 146F2/146R2 and the two EcoRI sites are also indicated.FIG. 15D shows the nucleotide sequences of six primer sets developedfrom the SalI-1461 bp fragment.

FIG. 16 shows the PCR amplification of WSBV and shrimp DNA specificfragments using DNA templates prepared from WSBV virions purified bysucrose gradient centrifugation. The WSBV specific primers 146F1 and146R1 which yield a 1447-bp PCR product were used for reactions in lanes2, 5, 8, and 11. The shrimp DNA specific primers 143F and 145R whichyield a 848-bp PCR product were used for reactions in lanes 3, 6, 9, and12. In lanes 4, 7, 10, and 13, all the primers 143F, 145R, 146F1 and146R1 were added together in each of the reactions. The PCR productswere analyzed on a 1% agarose gel. Lane 1, pGEM DNA size marker; lanes2-4, PCR products using DNA template extracted from virions purifiedfrom diseased shrimp #1 epidermis showing shrimp DNA and WSBV DNA band;lanes 5-7, PCR product using DNA template extracted from virionspurified from diseased shrimp #2 epidermis showing only WSBV DNA band;lanes 8-10, PCR product using DNA template extracted from virionspurified from diseased shrimp #2 muscle showing intense shrimp DNA andWSBV DNA band; lanes 11-13, PCR product using DNA template extractedfrom healthy shrimp showing only shrimp DNA band. The size of DNAmarkers is indicated in base pairs (bp).

FIG. 17 shows the PCR amplification of WSBV and shrimp DNA specificfragments using plasmid pms146 and DNA extracts from Penaeus monodonnaturally infected with WSBV as PCR DNA templates. The WSBV specificprimers 146F1 and 146R1 were used for reactions in lanes 2, 5, 8 and 11.The PCR product is a 1447-bp fragment. Internal primers specific to1447-kbp fragment, 146 F2 and 146 R2, were used for the reactions inlanes 3, 6, 9, and 12; they prime the amplification of a 941-kbpfragment. The shrimp DNA specific primers 143F and 145R were used forthe reactions in lanes 4, 7, and 10. They prime the amplification of a848-bp fragment. The amplification products were analyzed on a 1%agarose gel. Lane 1, pGEM DNA size marker); lanes 2-4, plasmid pms146;lanes 5-7, DNA extracts from naturally infected P. monodon; lanes 8-10,DNA extracts from naturally infected P. japonicus; lanes 11 and 12,template-free control reactions. The size of the DNA markers is given inbase pairs (bp).

FIG. 18 shows the PCR amplification of WSBV and shrimp DNA specificfragments using DNA templates prepared from Penaeus monodonexperimentally infected with WSBV. The primers 146F1 and 146 R1 whichyield a 1447-bp PCR product were used for the reaction. The PCR productswere analyzed on a 1% agarose gel. Lane 1, pGEN DNA size marker; lanes2-4, DNA extracts from 3 experimentally infected P. monodon; lanes 5-7,DNA extracts from healthy P. monodon of control group. The size of DNAmarkers is indicated in base pairs (bp).

FIG. 19. is a Dot hybridization of DNAs extracted from WSBV infected orhealthy Penaeus monodon with DIG-labeled 1447-kbp PCR product. The DNAsfrom 2 WSBV infected shrimp (1 and 2) and 2 healthy shrimp (3 and 4)were blotted in duplicate (A and B) onto the Hybond-N paper and probedwith DIG-labeled 1447-bp PCR product. The probe hybridized with the DNAsfrom the infected shrimp but not with the DNAs from the healthy shrimp.

FIG. 20 is a Southern hybridization of WSBV DNAs from the diseased P.monodon or P. japonicus with DIG-labeled 1447-bp PCR product. SalIdigested WSBV DNAs from P. monodon and P. japonicus were blotted ontothe Hybond-N paper and probed with DIG-labeled 1447-bp PCR product. Theprobe hybridized with a 1461-bp fragment of Sal I digested WSBV DNA fromeither shrimp source with equal visual intensity showing their closerelatedness. A: ethidium bromide-stained 0.8% agarose gel; B: theautoradiograph of the Southern blot of gel (A). Lane 1, pGEN DNA sizemarker; lane 2, genomic DNA SalI fragments of WSBV purified from P.monodon; lane 3 genomic DNA SalI fragments of WSBV purified from P.japonicus.

FIG. 21 shows the PCR amplication of WSBV DNA specific fragments usingprimer set 146F1/146F2 and DNA template prepared from arthropodscollected from epizootic areas. Lane 1: pGEN marker; lane 2: P. monodon;lane 3: P. japonicus; lane 4: crab; lane 5: copepoda; lame 6: insect(Family: Ephydridae); lane 7: positive control, DNA from known diseasedshrimp; and lane 8: negative control, sample without addition oftemplate.

DETAILED DESCRIPTION OF THE INVENTION

Outbreak of a disease causing serious finacial losses among populationsof cultured penaeid shrimps, including Penaeus monodon, P. japonicus andP. penicillatus in Taiwan is characterized by obvious white spot on thecarapace, appendages and the inside surface of the body. In order toidentify the causative agent of white spot syndrome in penaeid shrimps,electron microscope observations of diseased shrimps were conducted.Heathy juvenile kuruma shrimps (P. japonicus) were exposed by immersionto epidermal filtrate from diseased P. japonicus and P. monodon whichexhibited marked white spot signs. Challenge tests used this filtrate ondifferent sized kuruma shrimps.

A non-occluded rod-shaped virus particle was found by electronmicroscopy in the epidermis of both spontaneously and experimentallyinfected kuruma shrimps. Virions were enveloped, 330±20 nm in length and87±7 nm in diameter. These experimentally infected shrimps resembled thespontaneously affected ones. Direct inoculation of this virus-containingfiltrate into fish cell lines showed no cytopathic effect. Cumulativemortalities reached 100% within 5-7 days and were significantly affectedby catching and temperature stress.

The close resemblance in external signs and virus morphology betweenspontaneously diseased and experimentally infected shrimps indicatedthat the rod-shaped virus may be the main causative agent of the diseasein Taiwan characterized by white spot syndrome. For this reason, thisviral disease was proposed the name of “White Spot Syndrome” (W.S.S.).Further studies on the causative agent of W.S.S. (White Spot Syndromeassociated Virus) isolated from Penaeus monodon in order to know itstaxonomic position.

The causative viral agent was purified from diseased shrimp, Penaeusmonodon, with white spot syndrome. Negatively stained preparations showthat the virus is pleiomorphic. It is fusiform or rod-shaped. Innegatively stained preparations, the virion measures 70 to 150 nm at itsbroadest point and is 250 to 380 nm long. In some virions, a tail-likeprojection extends from one end. The capsid is apparently composed ofrings of subunits in a stacked series. The rings align perpendicularlyto the longitudinal axis of the capsid. The genome of the virus is adouble-stranded DNA molecule which produces at least 22 HindIIIfragments. The full length of the DNA is estimated to be longer than 150kbp. Based on the morphological characteristics and genomic structuresof the virus, it is confirmed that white spot syndrome associated virus(WSSV) is a member of genus NOB (Non-Occluded Baculovirus) of thesubfamily Nudibaculovirinae of Baculoviridae and the present isolate isdesignaetd as PmNOBIII, and as WSBV (Baculovirus associated with WhiteSpot syndrome) to indicate PmNOBIII related agents.

The WSBV may be closely related to hypodermal and hematopoietic necrosisbaculovirus (HHNBV) reported as the pathogen of the explosive epidemicdisease of prawn (EEDS) in China in 1993-1994 (Cai et al., J. Fish.China, 19: 112-117, 1995) and systematic ectodermal and mesodermalbaculovirus (SEMBV) of the black tiger prawn Penaeus monodon in Thailand(Wang et al., Dis. aquat. Org., in press, 1995; Wongteerasupaya et al.,Dis. aguat. Org., 21: 69-77, 1995).

The principal clinical sign of this new viral disease is the presence ofwhite spots on the exoskeleton and epidermis of the diseased shrimp withvaried sizes from barely visible to 3 mm in diameter. Histopathologicalstudy demonstrates that WSBV attacks most frequently the cuticularepidermis, as evidenced by the presence in these tissues of thedegenerated cells characterized by hypertrophied nuclei (Momoyama etal., Fish Pathol., 29: 141-148, 1994, Chou et al., Dis. aquat. Org., inpress, 1995; C. H. Wang et al., 1995). Thus, the white spot syndrome inpenaeid shrimp associated with non-occluded baculovirus can be said tobe a well-defined disease and in the prepresent studies we used anisolate of WSBV from P. monodon as the starting material to develop adiagnostic tool for the detection of WSBV in shrimps.

To develop a diagnostic tool for the detection of WSBV and related agentinfection in shrimps, the virions were purified from black tiger shrimpPenaeus monodon infected with WSBV. Extraction of viral genomic DNA frompurified virions was done by treating the virions with proteinase K andcetyltrimethyl-ammonium bromide (CTAB) followed by phenol-chloroformextraction and ethanol precipitation. A qualitative assessment wasperformed using polymerase chain reaction (PCR) on the viral DNA andprimers specific to shrimp genomic DNA for monitoring shrimp DNAcontamination in the viral genomic DNA preparations. A WSBV genomic DNAlibrary was constructed and based upon the sequence of the cloned WSBVDNA fragment, a WSBV specific primer set for PCR to detect the WSBVinfection in penaeid shrimps has been designed.

Samples which contained WSBV DNA yielded an evident amplificationproduct showing the expected mobility of a 1447-bp DNA fragment, whereasthe nucleic acids extracted from tissue samples from clinically healthyshrimp showed no Such DNA fragment, thereby confirming the specificityof the WSBV DNA specific primers designed in the present invention. ByPCR with the WSBV specific primer set, it has been demonstrated that thecausative agents of white spot syndrome in different shrimp species arein fact closely related. Other host organisms, including copepoda, crabsand insects are also tested for the presence of this new causative agentand the currently collected experimental data are positive. The resultsof this invention provide an effective diagnostic tool for screeningshrimp for WSBV infections, which may be extremely important inpreventing the further spread of this viral disease.

Materials and Methods

Shrimp. The healthy kuruma shrimps used for challenge tests wereobtained from a hatchery and a shrimp farm in southern Taiwan where noviral disease had been reported. All of the kuruma shrimps weremaintained at a temperature of 25-28° C. aquaria with aeration and fedan artificial, commercially obtained shrimp food twice daily. Diseasedshrimps of P. japonicus were collected from a culture farm in northernTaiwan, while samples of moribund penaeid shrimp, P. monodon (averageweight: 30 g) were collected from shrimp farms located in southernTaiwan in November 1994. The samples were examined by gross anatomy,light and electron microscopies for the confirmation of the diseaseusing the methodologies as described hereunder.

For light microscopy, both normal kuruma shrimps and individualsdisplaying marked white spot signs were preserved in Davison's fixative(Bell & Lightner 1988). After 48 h in Davison's fixative, specimens weretransferred to 50% ethanol, and then processed routinely for histologyto 5 μm paraffin wax sections, and stained routinely with hematoxylinand eosin (H & E).

For transmission electron macroscopy, sample of epidermis covering thegill chamber underneath the carapace was removed from naturally andexperimentally infected live kuruma shrimps, and immediately prefixed in2.5% glutaraldehyde in 0.1 M cold phosphate buffer solution (PBS, pH7.4) for 2 hr at 4° C. Subsequently, samples were washed several timesin cold PBS, and then postfixed in 1% osmium tetroxide for 3 hr at 4° C.The samples were dehydrated and embedded in Spurr's resin. Ultrathinsections were prepared on a Richert-jung Ultracut E Ultrotome, andstained with uranyl acetate and lead citrate. The sections were observedwith a HITACHI H-600 transmission electron microscope.

Challenge test. The epidermis from infected P. monodon was removed andhomogenized in brackish water at 4° C. in the ratio of 1:9. After beingcentrifuged at 8510×g (Sigma 2K15 rotor 12141) for 5 min, thesupernatant was filtered through a 0.45 μm membrane. The filtrate wascentrifuged at 14,549 xg (Sigma 2K15 rotor 12139) for 1.5 hr and tneresulting pellet was resuspended in sterilized brackish water beforebeing applied with negative staining. For negative staining, one drop ofsuspension was mixed with four drops of the mixture of 0.1% bovine serumalbumin and 2% phosphotungstic acid (1:2, pH 7.0). The mixture wasplaced on a 300 mesh grid for 30-60 sec and excess suspension wasremoved with filter paper. The preparation was allowed to dry beforebeing examined. Result was observed under a HITACHI H-600 transmissionelectron microscope.

Cytopathology assay. EPC (epithelioma papulosum cyprini), CHSE-214(chinook salmon embryo), FHM (fathead minnow) and SSE-5 (sockeye salmonembryo), cells were seeded in 24-well microplates. A filtrate was madefrom the epidermis of the diseased shrimps and was diluted from 1/20 to1/12500 in 5-fold dilutions. Diluted solutions were inoculated into thefour fish cell lines and these cells were observed over 2 weeks at anincubation temperature of 20° C.

An infection trial was performed using the filtrate of the epidermisfrom live or frozen naturally infected P. japonicus and P. monodon. Thefiltrate was diluted 500-750 times in brackish water in order to be usedas a waterborne inoculum. Two replicates of thirty-five one-month-oldjuvenile kuruma shrimps (mean weight 0.08 g) were immersed in thesediluted filtrates for 2 h. Two other populations were similarly exposed,either to the filtrate from healthy P. monodon epidermis or to Grace'sinsect medium. These served as controls. After inoculation, shrimps werekept in glass aquaria with aeration. Water temperature and salinity were25-28° C. and 25-30 ppt, respectively, throughout the experiment. Themortality was observed daily and the moribund shrimps were collected andexamined by transmission electron microscopy.

Purification, Genomic Structure and Taxonomic Position of WSBV

The purification of the virions of WSSV from P. monodon shrimp wascarried out as follows. The shrimps were first rinsed with cold 1×TEbuffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.6). The exoskeleton withunderlying epidermis taken from 1 to 5 live or frozen shrimp wasextracted with 20 ml cold extraction buffer (20 mM HEPES, 0.4 N NaCl, 1mM EDTA, 1 mM EGTA, 1 mM DTT, 2.5 mM phenylmethylsulfonyl fluoride, 1μg/ml leupeptin, 1.6 ug/ml pepstatin, 2 μg/ml aprotinin, 1 μg/mlbestatin), and then purified by centrifugation on linear 35 to 65% (W/W)sucrose gradient at 74,700 xg (Hitachi SRP 28SA rotor at 24,000 rpm) for60 min. The visible viral band in the midway of the gradient was removedand pelleted by centrifugation at 74,700 xg at 4° C. for 30 min. Thepellet was washed twice with cold 1×E buffer, resuspended with 300-500ml cold 1×TE buffer depending on the size of the pellet, and immediatelyused for viral DNA extraction. A small volume of purified virussuspensions was negatively stained with 2% phosphotungstic acid (PTA) atpH 7 for the ultrastructural studies of the virions.

The extraction of viral genomic DNA from gradient purified virions wasperformed by proteinase K and N-cetyl N,N,N-trimethylammonium bromide(CTAB) treatments followed by phenol-chloroform extraction and ethanolprecipitation (K. Wilson (1994), Preparation of genomic DNA frombacteria. Miniprep of bacterial genomic DNA. in Ausubel, F. M. et al.(eds.) Current Protocols in Molecular Biology, Vol. 1. Greene Pub.Assoc. and Wiley-Interscience, New York, p. 2.4.1-2.4.5).

The estimation of the viral genome size was done by restrictionendonuclease analysis. Viral DNAs were digested with HindIII, SalI andXhoI restriction endonucleases (Boehringer Mannheim Company).Restriction fragments were separated by electrophoresis in 0.8% agarosegel (9 cm×12 cm), with Tris-acetate buffer (0.04M Tris-acetate, 0.1 mMEDTA, pH 8.0) containing 0.5 μg/ml ethidium bromide. The 1-kilobase (kb)DNA ladder and lambda phage HindIII fragment marker (Life Technologies,Inc.) were used as the DNA size standard on the gel.

Development of Effective Diagnostic Tools

For the development of effective diagnostic tools, the construction ofWSBV genomic library was conducted by cloning “super pure” WSBV genomicDNA extracted from purified virions. In addition, the amplification ofselected DNA sequence by polymerase chain reaction (PCR) promises to bea powerful diagnostic tool for the identification of pathogens (Erlichet al., Nature 331: 461-462, 1988; Oste, C., Biotechniques 6: 162-167,1988). Based upon the sequences of the cloned WSBV DNA fragments, a WSBVspecific primer set for PCR has been designed.

I. WSBV genomic DNA library construction

A. Virus purification and extraction of viral DNA

The same batch of the frozen WSBV infected black tiger shrimp Penaeusmonodon, as used for the taxominic studies, was the source of the virus,and this strain of the WSBV is named as PmNOBIII (the third non-occludedbaculovirus reported for P. monodon) according to the criteria set forthin Francki et al. (Arch. Virol., 2: 1-450, 1991). The purification ofthe virions was carried out as described in the previous paragraphs. Theextraction of viral genomic DNA from purified virions was performed bytreating the virions with proteinase K and N-cetylN,N,N-trimethylammonium bromide (CTAB) followed by phenol-chloroformextraction and ethanol precipitation (Wilson (1994), supra). Briefly,the gradient-purified virions were incubated in TE buffer (10 mMTris-HCl, 1 mM EDTA, pH 7.6) containing 100 mM KCl, 1% SLS (N-laurylsarosine) and 0.2 mg/ml proteinase K at 65° C. for 3 hr. Afterincubation, 5 M NaCl was added to adjust the NaCl concentration of theDNA solution to 0.7 M. Next, 1/10 vol. CTAB/NaCl (10% CTAB in 0.7 MNaCl) was added slowly and mixed thoroughly before incubation at 65° C.for 10 min.

Following two extractions with an approximately equal volume ofchloroform/isoamyl alcohol and two extractions with an equal volume ofphenol/chloroform/isoamyl alcohol, the DNA was precipitated with twovolumes of absolute ethanol, and washed with cold 70% ethanol. The driedDNA pellet was dissolved in a suitable amount of 0.1×TE buffer at 65° C.for 30 min, and then stored at 4° C. until use.

B. Preparation of shrimp DNA for PCR as a control

The primers specific to shrimp genomic DNA for PCR were used to monitorshrimp DNA contamination in the WSBV genomic DNA preparations. For thispurpose, two primers were designed from the highly conserved regions of18S rRNA sequence of decapods, based on published sequences (Kim &Abele, J. Crust. Biol., 10, 1-13, 1990), a computerized data file(GenBank, National Institute of Health, Md., U.S.A.) and the sequencealignment analysis using PC/GENE program (Intelligenetics, Inc.). Bypairing the forward primer 143F (5′- TGC CTT ATC AGC TNT CGA TTG TAG-3′,where N represents G, A, T or C; SEQ ID NO:13) with a reverse primer145R (5′-TTC AGN TTT GCA ACC ATA CTT CCC-3′; SEQ ID NO:14), the shrimpDNA is expected to yield a PCR product of 848 bp corresponding tonucleotide sequences 352 to 1200 of 18S rRNA of P. aztecus.

The genomic DNAs extracted from the muscle of healthy P. monodon or P.japonicus were used as positive control for PCR. The deproteinizedgenomic DNA of the shrimp was prepared according to the method forpreparation of genomic DNA from mammalian tissue (Strauss, W M (1994)Preparation of genomic DNA from mammalian tissue. In Ausubel F M, BrentR, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K (Eds)Current Protocols in Molecular Biology, Vol. 1. Greene PublishingAssociates, Inc. and John Wiley and Sons, Inc., New York, p.2.2.1-2.2.3). Briefly, 200 mg muscle tissue excised from the abdomen ofthe shrimp was rapidly frozen in liquid nitrogen and crushed to a finepowder. The processed tissue was placed in 2.4 ml digestion buffer (100mM NaCl, 10 mM Tris-HCl, pH 8, 25 mM EDTA, pH 8, 0.5% sodium dodecylsulfate, 0.1 mg/ml proteinase K) and incubated at 65° C. for 12 to 18hr. The digest was deproteinized by successive phenol/chloroform/isoamylalcohol extractions, recovered by ethanol precipitation, and dried andresuspended in 0.1×TE buffer at 65° C. for 30 min, and then stored at 4°C. until use for PCR.

C. WSBV genomic DNA library construction

Two WSBV genomic libraries, PmNOBIII SalI (pms) and PmNOBIII HindIII(pmh) were contructed as set forth below. The WSBV genomic DNA withoutshrimp DNA contamination was digested with SalI or HindIII restrictionendonuclease (BRL, Life Technologies Inc.) at 37° C. for 3 hr in orderto obtain DNA fragments, and the fragments were then ligated into SalIor HindIII cleaved pUC 19 plasmid vector in the presence of T4 DNAligase at 16° C. overnight. The competent Escherichia coli DH 5α cellswere transformed with the resulting plasmids and plated onampicillin/isopropyl-β-D-thiogalactopyranoside(IPTG)/5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) agarplates. After using miniprep method to screen the whiteampicillin-resistant transformants for the presence of the appropriaterecombinant plasmids, both strands of the plasmid inserts were sequencedwith double-stranded DNA templates using a Sequenase kit (United StatesBiochemical Corp.) with M13/pUC Sequencing Primers (GIBCO BRL LifeTechnologies Inc.), and subsequently, specific internal primers.Recombinant plasmids were isolated from transformants and screened forthe presence of the insert by SalI or HindIII digestion. The size of theinserts were listed in Table 1.

II. Amplification of WSBV DNA fragment from DNA extracted from purifiedWSBV virions

Oligonucleotide primers (146F and 146R) are used for the amplificationof WSBV DNA fragments. Primers 146F and 146R are designed on the basisof the DNA sequence of a cloned WSBV 1461-bp SalI DNA fragment inrecombinant plasmid pms146 and there have been established 6 primer setsas shown in FIG. 15D. The primer set of 146R1 and 146F1 have thefollowing nucleotide sequences: 146R1, 5′-AA TGC GGG TGT AAT GTT CTT ACGA-3′(SEQ ID NO:4); 146F5′-AAC TTC AGC CTA TCT AG-3′(SEQ ID NO:3). Withthis primer set, a 1447-bp fragment is expected to be amplified fromWSBV genomic DNA. The internal primer set, 146R2, 5′-TAC GGC AGC TGC TGCACC TTG T-3′(SEQ ID NO:6), and 146F2, 5′-GTA ACT GCC CCT TCC ATC TCCA-3′(SEQ ID NO:5) are used to confirm that the amplified fragment isindeed from the WSBV 941-bp SalI DNA fragment.

The deproteinized DNA samples extracted from purified WSBV virions andfrom the muscle of the healthy shrimp were used as DNA templates for theevaluation of the specificity of the primers by PCR.

III. Amplification of WSBV DNA fragment from DNA extracted from tissuesof shrimp naturally and experimentally infected with WSBV

The diseased shrimps consisted of shrimp naturally and experimentallyinfected with WSBV. For experimental infection, the healthy shrimp(average body weight: 0.5 gm) were infected with WSBV using the methoddescribed in the preceding paragraphs. Five days after infection, theDNAs were extracted from three experimentally infected shrimp and threehealthy shrimp and checked by PCR with the use of WSBV specific primers(146F1 and 146R1) and shrimp DNA specific primers (143F and 145R).

IV. PCR amplification and analysis of products

The deproteinized DNA samples used for amplification totaled 9.1-0.3 μgin a 100 μl reaction mixture containing 10 mM Tris-HCl, pH 9 at 25° C.,50 mM KC1, 1.5 mM MgCl2, 0.1% Triton X-100, 200 μM each of dNTP, 100pmol each of primer, 2.5 units of Taq DNA Polymerase (Promega). Theamplification was performed in a AG-9600 Thermal Station (BiotronicsCorp.) for one cycle of 94° C. for 4 min, 55° C. for 1 min, 72° C. for 3min; 39 cycles of 94° C. for 1 min, 55° C. for 1 min, 72° C. for 3 min,plus a final 5 min extension at 72° C. after 40 cycles. Controlreactions containing no template DNAs were run for all PCR reactions. Insome PCR reactions, controls also consisted of reaction mixtures withDNA extracts from healthy shrimp. The PCR products were analyzed in 1%agarose gels containing ethidium bromide at a concentration of 0.5μg/ml, and visualized under an ultraviolet transillumination.

V. Dot hybridization of DNAs extracted from WSBV infected or healthy P.monodon with DIG-labeled 1447-bp PCR product

The DNAs extracted from WSBV infected or healthy P. monodon were spottedonto Hybond-N paper (Amersham) using a 96-well dot-blot vacuumfiltration manifold apparatus (Schleicher and Schuell, Inc.). The blotswere air dried and denatured in 1.5 M NaCl, 0.5 N NaOH for 10 min, andthen neutralized in 1.5 M NaCl, 1 M Tris, pH 7.4 for 10 min. The blotswere used for hybridization with a DIG-labeled 1447-bp PCR productfollowing the standard molecular cloning techniques (Sambrook J, FritschE F, Maniatis T (1989,) Molecular Cloning: A Laboratory Manual, 2nd.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The dotblot was hybridized at 37° C. for 16 hr with the DIG-labeled probe,after prehybridization at 37° C. for 12 hr in 50% formamide, 5X SSC, 1mM EDTA, 50 mM Tris (pH 8), 5X Denhardt's reagent (0.1% Ficoll-400, 0.1%polyvinyl pyrrolidone, 0.1% BSA). The 1447-bp PCR product was used as atemplate to prepare probe using the random primer method (BoehringerMannheim). After hybridization, the detection of the DIG-labelednucleotides in blots was accomplished with a chemiluminescent reactionby using the DIG Luminescent Detection Kit (Boehringer Mannheim). Theblot was exposed to Kodak XAR-5 film at 37° C. for 15-30 min to recordthe chemiluminescent signal.

VI. Southern hybridization of WSBV DNAs from the diseased

P. monodon or P. japonicus with DIG-labeled 1447-bp PCR product

Southern blot hybridization was performed to localized the 1447-bp PCRproduct within the genomic DNA of WSBV purified from the diseased P.monodon or P. japonicus with white spot syndrome. For this purpose, 200ng genomic DNA of WSBV isolated from the diseased shrimp was digestedwith SalI, and then electrophoretically separated in 0.8% agarose gel.After acid (0.25 N HCl) depurination and alkali (1.5 M NaCl-0.5 N NaOH)denaturation of the DNA, the gel was neutralized with 1 M Tris (pH 7.4)and 1.5 M NaCl, and subsequently transferred to a Hybond-N nylonmembrane using a vacuum transfer unit (Hoefer TE 80) for 60 min. The 20XSSC (3 M NaCl, 1.5 M Sodium Citrate) was used as transfer buffer(Sambrook et al. (1989), supra). The blot was used for hybridizationwith a DIG-labeled 1447-bp PCR product.

VII. Detection of WSBV in Arthropods Collected from Epizootic Areas

The DNA templates prepared from arthropods collected from epizooticareas were used for PCR with 146R1 and 146F1 primers for the detectionof WSBV in the tested organisms.

RESULTS

A. Histopathological studies

Outbreaks of W.S.S. amongst penaeid shrimps is evidently not a confined,local problem anymore. It has already brought the cultured shrimpindustry in Asia to a critical condition. In order to classify thecausative virus more clearly and develop a quick diagnostic method,further studies on the physicochemical characterization of this agent isconducted.

The main clinical signs of the disease in Penaeus monodon were the whitespots on the exoskeleton (FIG. 1). The white spots were particularlyobvious on the carapace removed from the diseased shrimp, and werereadily observed even on the carapaces from lightly infected animals.Histopathological study demonstrates that epidermis of the diseasedshrimp was attacked by viral agent evidenced by the presence in thistissue of the degenerated cells characterized by hypertrophied nucleiwith inclusions (FIG. 2).

Ultrathin sections of the underlying epidermis of the cuticle fromshrimp with white spot syndrome viewed under the electron microscoperevealed numerous non-occluded baculo-like virus particles in thenecrotic areas. The hypertrophied nuclei filled with virions were alsoreadily seen (FIG. 3). The virus particles were 330±20 nm in length and87±7 nm in diameter (n=30). The electron-dense central core of the virusparticle is nucleocapsid, approximately 220×70 nm in size. No differencein virion morphology between spontaneouly diseased and experimentallyinfected shrimp was recognized (FIG. 4).

B. Negative staining and cytotoxicity assay of the filtrate forchallenge test

The result of negative staining of the pellet from the filtrate ofdiseased P. monodon epidermis is shown in FIG. 5. Virus particles withrod-shape morphology can be seen. These are similar to the virusparticles observed in ultrathin sections of spontaneously diseasedshrimps. No bacteria were observed.

Cytopathic effect (CPE) was not found in any of the four tested fishcell lines; the filtrate which was used as waterborne inoculum had nocytotoxicity.

C. Challenge test

Healthy shrimps were exposed to epidermal filtrate from disesed P.japonicus and P. monodon which exhibited marked white spot symptoms.These experimentally infected shrimps resembled the spontaneouslyaffected ones (FIG. 6) and cumulative mortalities reached 100% within5-7 days (FIG. 7), while no shrimp died in the control groups.

Further, the inoculum was highly pathogenic to the smallest shrimpstested (mean weight of 0.08 g) and all these shrimps died within 5 days;only 35% cumulative mortality was found in the 0.16 g-sized shrimp groupafter 7 days although mortality reached 100% in 12 days; and 10%mortality was observed in the group of largest shrimp (mean weight of0.26 g) within 2 weeks. No shrimp died in the control groups.

D. Purification, Genomic Structure and Taxonomic Position of WSBV

The purified virions were fusiform or rod-shaped with bluntly roundedends. In negatively stained preparations, the virion was 70 to 150 nm atits broadest point, and was 250 to 380 nm long, which is usually 10%larger than in ultrathin sections. In some virions, a tail-likeprojection extending from one end was observed (FIG. 8). Thenon-enveloped nucleocapsids were normally 58 to 67 nm in diameter and330 to 350 nm long. The capsid components formed parallelcross-striations (FIG. 9). Thus, the capsid seemed to be composed ofrings of subunits in a stacked series. The thickness of the rings (20nm) was very constant and the rings were perpendicular to thelongitudinal axis of the capsid. In term of virus morphology, WSSVresembles SEMBV (Systemic Ectodermal and Mesodermal Baculovirus) anddiffers from BMN (Baculoviral Mid-gut Gland Necrosis Virus) and PmSNPV(Penaeus monodon Single Nucleocapsid Nuclear Polyhedrosis virus=MBV)(Mari et al., Dis. aquat. Org. 16: 207-215, 1993; Sano et al.,Helgolander Meeresunters. 37: 255-264, 1984; Wongteerasupaya et al.,Dis. aquat. Org. 21: 69-77, 1995). However, the main clinical sign ofwhite spot caused by WSSV was not described in the SEMBV infectedshrimp. To date, it is difficult to guess the relatedness between WSSVand SEMBV.

A single DNA molecule was extracted from purified virions of WSBV (FIG.10). The genomic DNA of WSBV digested with HindIII, SalI and XhoIrestriction endonucleases was shown in FIG. 11. The genomic DNA of WSSVdigested with HindIII restriction endonuclease produced, in the agarosegel, at least 22 fragments of approximate sizes: 19.4, 16.9, 14.9, 12.5,10.0, 9.6, 8.4, 8.0, 7.3, 6.1, 5.5, 4.8 , 4.3, 3.9, 3.6, 3.3, 3.0, 2.5,2.0, 1.6, 1.4, and 1.1 kbp, respectively. The fragments smaller than 1kbp have run over the gel if they exist. The length of WSBV DNA wasestimated to be longer than 150 kbp, which falls within the size rangeof 90-230 kbp found in insect baculoviruses (Francki et al., Arch.Virol., suppl. 2: 1-450, 1991).

Based on the morphological characteristics and genomic structure, WSSVis classified as the genus Non-Occluded Baculovirus (NOB) of thesubfamily Nudibaculovirinae of Baculoviridae (Francki et al. (1991),supra) and the isolate was named PmNOBIII, as the third non-occludedbaculovirus reported for P. monodon (D. V. Lightner, Boca Raton, p.393-486, 1993; Wongteerasupaya et al (1995), supra). The present virusisolate PmNOBIII was deposited in the China Center for Type CultureCollection (CCTCC) of the People's Republic of China on Jan. 11, 1996(Accession Number CCTCC-V96001) under the Budapest Treaty. It is alsoproposed to use WSBV (Baculovirus associated with White-spot Syndrome)to indicate the PmNOBIII related agents.

E. Development of Effective Diagnostic Tools for WSBV Infection

I. WSBV genomic DNA library construction

a) Virus purification and extraction of viral DNA

Typical rod-shaped virions of WSBV were readily observed afterconcentration and purification by sucrose gradient centrifugation. Thesevirions were used to extract the viral DNA.

The amplification of shrimp DNA using PCR and primers specific to 18SrRNA reliably resulted in a predicted 848-bp DNA fragment (FIG. 12).This provided a simple and highly sensitive method for detecting smallamounts of shrimp DNA and was subsequently used to monitor shrimp DNAcontamination in WSBV genomic DNA preparations for library construction.The PCR analysis shown in FIG. 13 indicates that host DNA contaminationwas detected in most WSBV genomic DNA preparations. However, a fewsamples of WSBV genomic DNA extracted from purified virion preparationswere virtually free of contaminating host DNA. An example is shown inFIG. 13, lane 3.

b) Genomic DNA library construction

Two WSBV genomic libraries, PmNOBIII SalI (pms) and PmNOBIII HindIII(pmh) were contructed with use of SalI or HindIII restrictionendonuclease (BRL, Life Technologies Inc.) and pUC 19 plasmid vector.

For example, the SalI digested WSBV DNA was checked by electrophoresinga 5-μl aliquot in a 0.8% agarose gel containing ethidium bromide. TheWSBV genomic DNA was completely digested with SalI restrictionendonuclease (FIG. 14). From the same batch of digested DNA, a 20-μlaliquot was used for library construction.

Recombinant plasmids isolated from transformants were screened by SalIor HindIII digestion, among which 592 clones (pms1-pms592) from pmslibrary and 410 clones (pmh1-pmh245 and pmh419-pmh584) from pmh librarywere screened for the presence of the insert by SalI or HindIIIdigestion. The size of the inserts varied from 15 kbp to less than 100bp as shown in Table 1. These libraries provide an abundant supply ofWSBV DNA, enabling further study of the molecular biology of the virusand development of nucleic acid and immunological diagnostic kits.

II. Amplification of WSBV DNA fragment from deproteinized DNA extractedfrom purified virions

On the basis of the obtained-DNA sequences (data not shown) of WSBV SalIDNA fragments, several primer sets were designed and evaluated by PCRfor their ability to identify the WSBV in infected tissues.

FIG. 15A displays a diagram of the SalI-1461 bp DNA fragment cloned inplasmid pms146 and FIG. 15B shows the locations of the primers, whichare used for PCR amplication, in the SalI-1461 bp DNA fragment. The146F1 and 146R1 prime the amplification of a 1447-bp fragment, while146F2 and 146R2 prime the amplification of a 941-bp fragment. Thepositions of two EcoRI sites in SalI 1461 bp DNA fragment are alsoindicated. FIG. 15C shows the detailed nucleotide sequence of theSalI-1461 bp fragment, in which the locations of the two primer sets146F1/146R1 and 146F2/146R2 and the two EcoRI sites are also indicated.FIG. 15D shows the nucleotide sequences of six primer sets developedfrom the SalI-1461 bp fragment. Among them, the primer set 146F/146R1gave a consistent and an efficient amplification of WSBV DNA but not ofshrimp DNA. This primer set was then chosen for subsequent parts of thisstudy.

FIG. 16 shows the results of amplification using purified WSBV genomicDNA as PCR template, and the primer sets either specific to WSBV DNA orto shrimp DNA. The reactions analyzed in FIG. 16, lanes 2, 5 and 8represent amplification using WSBV DNA primer set 146F1-146R1 and threeindependent WSBV DNA preparations, and the results demonstrate thepresence of a relatively large amount of WSBV genomic DNA in the threetested samples, as evidenced by an intense 1447-bp PCR product in theselanes. At least one of the WSBV DNA preparations is free from shrimp DNAcontamination, as evidenced by the absence of detectable PCR product ofshrimp DNA in FIG. 16, lane 6. The WSBV primer set 146F1/146R1 andshrimp DNA primer set 143F/145R were used simultaneously in a reactionmixture for demonstrating approximately the proportion of WSBV DNA intemplate DNAs.

The data presented in FIG. 16 demonstrate that WSBV specific DNAfragment was detected as a major band in three independent WSBVpreparations (lanes 4, 7 and 10) while the shrimp DNA was detected intwo of three WSBV DNA preparations (lanes 4 and 10). Thus template DNAscontained varying proportions of shrimp DNA and WSBV DNA. It is alsoclear that in spite of contamination with shrimp DNA, a large proportionof the DNAs extracted from WSBV virions purified by sucrose gradientcentrifugation is WSBV DNA. Meanwhile, reaction mixtures with totalnucleic acid extracted from tissues from clinically healthy shrimp andWSBV DNA specific primer set 146F1/146R1 were consistently negative(FIG. 16, lane 11), thus demonstrating the specificity of this primerset.

III. Amplification of WSBV DNA fragment from DNA extracted from theshrimp tissues naturally and experimentally infected with WSBV

FIG. 17 shows the amplification results using plasmid pms146 DNA and theDNA extracted from the tissues of P. monodon and P. japonicus natuallyinfected with WSBV as DNA templates. The DNA templates were amplifiedusing either the WSBV-specific primer set 146F1/146R1 or shrimpDNA-specific primer set 143F/145R. The 1447-bp PCR product, comigratingwith DNA amplified from pure plasmid pms146 DNA, demonstrates thepresence of WSBV DNA in the total nucleic acid extracted from all thenaturally infected shrimp. Examples are shown in FIG. 17, lanes 2, 5 and8.

Using the internal primer set 146F2/146R2, 10 μl of these products werereamplified to yield a PCR product with the expected size of 941 bp(FIG. 16, lanes 3, 6 and 9). The results confirm the identity betweenamplification product and template. Shrimp DNA was amplified veryefficiently using shrimp DNA specific primer set 143F/145R as shown inFIG. 17, lanes 7 and 10. The results presented in FIG. 17, lanes 5 to 10demonstrate that WSBV DNA could be detected with the use of WSBV DNAspecific primer sets 146F1/146R1 and 146F2/146R2 in the presence of alarge excess of shrimp genomic DNA.

FIG. 18 shows the amplication result using DNA extracted from tissues ofP. monodon experimentally infected with WSBV as DNA templates for PCRusing primer set 146F/146R1. Amplification of the expected 1447-bpfragment is evident for all the experimentally infected shrimp. Noamplification product at 1447 bp was present for healthy shrimp fromcontrol group.

IV. Dot hybridization of DNAs extracted from WSBV infected or healthy P.monodon with DIG-labeled 1447-bp PCR product

The results of dot hybridization demonstrate that the PCR producthybridized with DNAs extracted from WSBV infected shrimp, but did nothybridize with DNAs extracted from healthy shrimp. (FIG. 19). Theresults demonstrate the specificity of the 1447-bp PCR product.

V. Southern hybridization of WSBV DNAs from the diseased P. monodon orP. japonicus with DIG-labeled 1447-bp PCR product

In order to localize the 1447-bp PCR product within the WSBV genomicDNA, Southern hybridization of WSBV genomic DNA SalI fragments wasperformed using DIG-labeled 1447-bp PCR product as a probe. The resultsdemonstrate that 1447-bp PCR product hybridized specifically with a WSBVgenomic DNA SalI fragment of 1461 bp (FIG. 20). Both 1461 bp SalIfragments of WSBV genomic DNAs prepared respectively from P. monodon andP. japonicus were found to be positive with the probe.

VI. Detection of WSBV in Arthropods Collected from Epizootic Areas

Among tested organisms, P. monodon, P. japonicus, crabs, copepoda andinsect (Family: Ephydridae) gave WSBV positive results (FIG. 21).

DISCUSSION

The diseased shrimps have obvious white spots on the carapace,appendages and the inside surface of the body, and also display signs oflethargy and reddish coloration of the hepatopancreas. Vibriosis, virusinfection, poor environmental management and nutrient imbalance have allbeen conjectured to be the possible cause for these outbreaks. Based onelectron microscope observation, however, a rod-shaped virus wasconsidered to be the main causative agent. In the present study, thepathogenicity of a pathogenic virus from diseased P. japonicus and P.monodon with white spot syndrome was investigated. Close resemblance inwhite spot signs and virus morphology between spontaneously diseased andexperimentally infected shrimps demonstrated that this virus is indeedthe causative agent of the outbreak. The virus is highly pathogenic andconstitutes a threat to shrimp. Information pilot studies in whichdiseased shrimps were fed to healthy specimens suggest that the virusmay be transmitted orally as well as via water.

In addition to WSBV, a variety of baculoviruses has been reported toinfect decapod crustaceans since the first report by Couch (Nature, 247(5438):229-231, 1974; J. Invertebr. Pathol., 24: 311-331, 1974) and someof them cause mass mortality of the diseased animals (Lightner & Redman,J. Invertebr..Pathol., 38: 299-302, 1981; Sano et al., Fish Pathol., 15:185-191, 1981; Lester et al., Dis. aquat. Org., 3: 217-219, 1987;Johnson P. T., Dis. aquat. Org., 5: 111-122, 1988; Johnson & Lightner,Dis. aquat. Org., 5: 123-141, 1988; Bruce et al., J. Virol. mehods, 34:245-254, 1991; Chang et al., Fish Pathol., 27 (3): 127-130, 1992; Changet al., J. Invertebr. Pathol., 62: 116-120, 1993; Mari et al., Dis.aquat. Org., 16: 207-215, 1993, Wongteerasupara et at., Dis. aquat.Org., 21: 69-77, 1995). These viruses are morphologically similar, andmost researchers agree that the structure of the viral genome shouldbecome the much needed reference for determining the taxonomic positionof crustacean baculoviruses. The development of rapid and reliablediagnostic tools using molecular approaches will be useful not only forthe identification and comparative studies of the viruses but also forthe screening of carriers in shrimp larvae and parental spawners. Inview of these points the present researches are focused on the WSBVgenomic structure and on the development of rapid and sensitivediagnostic tools.

In experiment, shrimp DNA specific primers are used in severalassessments. The aims of the use of shrimp DNA specific primer set inthe present study were (i) to assess the purity of WSBV genomic DNApreparations, (ii) to evaluate nucleic acid extraction procedures foryielding amplificable DNA template, and (iii) to estimate approximatelythe proportion of the shrimp DNA and WSBV DNA in template DNAs preparedfrom total nucleic acids of the infected tissues. Attempts have beenmade in our laboratory to purify WSBV virions from various tissuesincluding epidermis, muscle and gills. From these virions we obtainedWSBV DNA of varied purity as assessed by shrimp DNA specific primers.Examples of these assessments are shown in FIGS. 13 and 17. The nucleicacids extracted from muscle tissues yielded a great quantity of WSBVDNA, but were heavily contaminated with shrimp DNA (FIG. 16, lane 9).The virions purified from heavily infected epidermal cells underneaththe exoskeleton are good starting materials for extracting “super pure”WSBV genomic DNA (FIG. 16 lanes 2 and 5). By using the shrimp DNAspecific primers and PCR, for the first time a tool is available toassess the extent of the shrimp DNA contamination in shrimp virusgenomic DNA preparations.

Using the WSBV DNA specific primers, all the purified WSBV genomic DNAsamples consistently yielded an evident amplification product showingthe expected mobility of a 1447-kbp DNA fragment. The nucleic acidsextracted from tissues of naturally diseased shrimp-with white spotsyndrome and from shrimp experimentally infected with WSBV alsoconsistently gave PCR products of the same size. The nucleic acidsextracted from the tissues of clinically healthy shrimp showed nopositive results. These results demonstrate the specificity of the WSBVDNA specific primers designed in the present study. In addition, the1447-bp PCR product can be used to prepared WSBV specific nucleic acidprobe for detecting WSBV infection in shrimp using dot blothybridization as shown in FIG. 19. Practically, the present studiesprovide three effective diagnostic tools for screening of the WSBVinfection in penaeid shrimps as shown in FIGS. 17, 19 and 20.

With PCR (FIG. 17) and Southern hybridization (FIG. 20), we havedemonstrated that the causative agents of white spot syndrome ofdifferent shrimp species are in fact closely related. Screening for theWSBV infection in shrimp should be undertaken immediately in order toprevent this viral disease from spreading further. On the other hand,the PCR diagnostic techniques for WSBV developed in the present studyprovide effective tools for the comparative studies on the shrimpnon-occluded baculoviruses such as Japan' RV-PJ (Inouye et al (1994),supra), China' HHNBV ( Cai et al., J. Fish. China, 19: 112-117, 1995),Thailand' SEMBV (Wongteerasupaya et al. (1995), supra), the present WSBVisolate PmNOBIII and other crustacean non-occluded baculoviruses.

From the above teachings, it is apparent that various modifications andvariations can be made without departing from the spirit and scope ofthe present invention. It is therefore to be understood that thisinvention may be practiced otherwise than as specifically described.

TABLE 1 The insert size (in kilo base pair; kb) of clones in PmNOBIIISalI (pms) and PmNOBIII HindIII (pmh) libraries. PmNOB SalI library(pms) PmNOB HindIII library (pmh) clone no. insert size (kb) clone no.insert size (kb) pms1 0-0.1 pmh1 0.3 pms2 0-0.1 pmh2 7-8 pms3 0-0.1 pmh34 pms4 0-0.1 pmh4 0-0.1 pms5 0-0.1 pmh5 0-0.1 pms6 0-0.1 pmh6 0-0.1 pms74 pmh7 5 pms8 ? pmh8 3 pms9 0-0.1 pmh9 ? pms10 0-0.1 pmh10 9 pms11 0-0.1pmh11 7-8 pms12 0-0.1 pmh12 3.5 pms13 0-0.1 pmh13 9-23 pms14 0-0.1 pmh142.2 pms15 0-0.1 pmh15 4 pms16 0-0.1 pmh16 6 pms17 3 pmh17 7 pms18 0-0.1pmh18 6 pms19 ? pmh19 7 pms20 ? pmh20 1.5 pms21 0-0.1 pmh21 1.7 pms22 ?pmh22 8 pms23 0-0.1 pmh23 2.2 pms24 ? pmh24 0-0.1 pms25 ? pmh25 0-0.1pms26 0-0.1 pmh26 0-0.1 pms27 0-0.1 pmh27 2.2 pms28 0-0.1 pmh28 6 pms290-0.1 pmh29 0-0.1 pms30 0-0.1 pmh30 5 pms31 0-0.1 pmh31 4 pms32 0-0.1pmh32 9 pms33 0-0.1 pmh33 6 pms34 0-0.1 pmh34 7-8 pms35 0-0.1 pmh35 0.5pms36 0-0.1 pmh36 ? pms37 0-0.1 pmh37 3.5 pms38 0-0.1 pmh38 1.5 pms390-0.1 pmh39 4 pms40 4 pmh40 6 pms41 0-0.1 pmh41 6 pms42 0-0.1 pmh42 1.5pms43 0-0.1 pmh43 1 pms44 ? pmh44 0-0.1 pms45 0-0.1 pmh45 6 pms46 0-0.1pmh46 ? pms47 0-0.1 pmh47 1.7 pms48 0-0.1 pmh48 3 pms49 3 pmh49 8 pms50? pmh50 2.2 pms51 ? pmh51 0-0.1 pms52 2 pmh52 4 pms53 0-0.1 pmh53 0-0.1pms54 5-6 pmh54 0-0.1 pms55 0-0.1 pmh55 8 pms56 2 pmh56 3.5 pms57 0.5pmh57 ? pms58 0-0.1 pmh58 5 pms59 ? pmh59 3.5 pms60 4-5 pmh60 0-0.1pms61 ? pmh61 3.2 pms62 0-0.1 pmh62 2 pms63 0.5-1 pmh63 0-0.1 pms640-0.1 pmh64 4 pms65 2-3 pmh65 6 pms66 1.5 pmh66 3.5 pms67 0-0.1 pmh671.5 pms68 0.5 pmh68 ? pms69 0.3 pmh69 4.4 pms70 0.2 pmh70 7 pms71 ?pmh71 ? pms72 0.5 pmh72 ? pms73 ? pmh73 7 pms74 0-0.1 pmh74 ? pms750-0.1 pmh75 ? pms76 5-6 pmh76 ? pms77 0-0.1 pmh77 ? pms78 0.2 pmh780-0.1 pms79 ? pmh79 ? pms80 0.3 pmh80 pms81 ? pmh81 ? pms82 0.5 pmh82 ?pms83 3 pmh83 ? pms84 ? pmh84 4.4 pms85 ? pmh85 ? pms86 3 pmh86 ? pms87? pmh87 ? pms88 ? pmh88 ? pms89 ? pmh89 0-0.1 pms90 ? pmh90 ? pms91 0.3pmh91 ? pms92 6 pmh92 ? pms93 0.2 pmh93 9 pms94 9 pmh94 8 pms95 0-0.1pmh95 ? pms96 ? pmh96 ? pms97 0.3 pmh97 ? pms98 4 pmh98 1.5 pms99 0-0.1pmh99 ? pms100 6 pmh100 ? pms101 ? pmh101 0-0.1 pms102 0.3 pmh102 7pms103 7-8 pmh103 1.5 pms104 ? pmh104 1 pms105 ? pmh105 9 pms106 2pmh106 ? pms107 0-0.1 pmh107 2.2 pms108 0-0.1 pmh108 0.5 pms109 3-4pmh109 0-0.1 pms110 3 pmh110 0-0.1 pms111 3-4 pmh111 ? pms112 1.5 pmh112? pms113 2 pmh113 ? pms114 ? pmh114 ? pms115 8 pmh115 2.2 pms116 2pmh116 ? pms117 9 pmh117 ? pms118 ? pmh118 ? pms119 0-0.1 pmh119 9-23pms120 4 pmh120 2.5 pms121 0-0.1 pmh121 ? pms122 3 pmh122 ? pms123 0-0.1pmh123 ? pms124 4 pmh124 ? pms125 0-0.1 pmh125 ? pms126 7 pmh126 ?pms127 ? pmh127 ? pms128 0-0.1 pmh128 ? pms129 ? pmh129 ? pms130 3pmh130 ? pms131 0-0.1 pmh131 ? pms132 ? pmh132 ? pms133 ? pmh133 ?pms134 7-8 pmh134 ? pms135 0-0.1 pmh135 ? pms136 ? pmh136 ? pms137 ?pmh137 6 pms138 ? pmh138 2 pms139 ? pmh139 ? pms140 ? pmh140 0-0.1pms141 2 pmh141 ? pms142 ? pmh142 4 pms143 ? pmh143 0-0.1 pms144 ?pmh144 ? pms145 ? pmh145 0-0.1 pms146 1.5 pmh146 ? pms147 ? pmh147 ?pms148 ? pmh148 ? pms149 ? pmh149 0-0.1 pms150 ? pmh150 ? pms151 0.2pmh151 7 pms152 ? pmh152 2.2 pms153 ? pmh153 ? pms154 ? pmh154 ? pms155? pmh155 ? pms156 ? pmh156 ? pms157 ? pmh157 ? pms158 ? pmk158 ? pms1590.2 pmh159 0-0.1 pms160 1 pmh160 ? pms161 ? pmh161 ? pms162 0.3 pmh1620-0.1 pms163 0-0.1 pnih163 0-0.1 pms164 4 pmh164 5 pms165 0-0.1 pmh165 ?pms166 0-0.1 pmh166 ? pms167 0.2 pmh167 1 pms168 1.5 pmh168 7 pms1690-0.1 pmh169 ? pms170 0.5 pmh170 1 pms171 0-0.1 pmh171 ? pms172 0-0.1pmh172 ? pms173 0.3 pmh173 ? pms174 0-0.1 pmh174 ? pms175 0-0.1 pmh1751.3 pms176 0-0.1 pmh176 ? pms177 0-0.1 pmh177 ? pms178 0-0.1 pmh178 6pms179 0-0.1 pmh179 3 pms180 1.5 pmh180 3 pms181 4 pmh181 ? pms182 0.5pmh182 9 pms153 0.5 pmh183 0-0.1 pms184 0-0.1 pmh184 ? pms185 0.5 pmh1854.4 pms186 0-0.1 pmh186 0-0.1 pms187 0-0.1 pmh187 9 pms188 0-0.1 pmh1881 pms189 2 pmh189 ? pms190 0-0.1 pmh190 ? pms191 0-0.1 pmh191 ? pms1920-0.1 pmh192 ? pms193 0-0.1 pmh193 ? pms194 0-0.1 pmh194 ? pms195 >9pmh195 ? pms196 0-0.1 pmh196 1.5 pms197 1.5 pmh197 ? pms198 0-0.1 pmh198? pms199 ? pmh199 ? pms200 ? pmh200 1.7 pms201 0-0.1 pmh201 1 pms202 ?pmh202 9 pms203 1.5 pmh203 6 pms204 ? pmh204 4.5 pms205 ? pmh205 7pms206 ? pmh206 7 pms207 0.3 pmh207 7 pms208 ? pmh208 8 pms209 0.3pmh209 7 pms210 0.3 pmh210 4.5 pms211 ? pmh211 3.2 pms212 ? pmh212 3.2pms213 0-0.1 pmh213 4.4 pms214 1.5 pmh214 1.5 pms215 ? pmh215 3 pms216 4pmh216 4.5 pms217 ? pmh217 3 pms218 1 pmh218 7 pms219 0-0.1 pmh219 3pms220 ? pmh220 3.2 pms221 ? pmh221 2.2 pms222 ? pmh222 2.2 pms223 ?pmh223 2.4 pms224 ? pmh224 1.7 pms225 0.5 pmh225 4.2 pms226 ? pmh226 2.2pms227 0.3 pmh227 4.2 pms228 0.3 pmh228 4 pms229 ? pmh229 1.5 pms2300-0.1 pmh230 2.2 pms231 7-8 pmh231 2.2 pms232 0.3 pmh232 4.2 pms233 0.3pmh233 2.2 pms234 0-0.1 pmh234 1.5 pms235 0.5 pmh235 4.2 pms236 0.4pmh236 2.2 pms237 0.3 pmh237 2.2 pms238 1.5 pmh238 4.4 pms239 3 pmh239 3pms240 1 pmh240 3 pms241 0.5 pmh241 2.6 pms242 0.3 pmh242 5 pms243 ?pmh243 3 pms244 0-0.1 pmh244 2.5 pms245 0.5 pmh245 3.2 pms246 0-0.1pmh246 pms247 ? pmh247 pms248 0.5 pmh248 pms249 7-8 pmh249 pms250 4-5pmh250 pms251 0.3 pmh251 pms252 0-0.1 pmh252 pms253 0.2 pmh253 pms2540-0.1 pmh254 pms255 0.3 pmh255 pms256 0.2 pmh256 pms257 ? pmh257 pms2580.5 pmh258 pms259 0.4 pmh259 pms260 0-0.1 pmh260 pms261 ? pmh261 pms2620.3 pmh262 pms263 ? pmh263 pms264 2.5 pmh264 pms265 ? pmh265 pms2660-0.1 pmh266 pms267 7-8 pmh267 pms268 0.2 pmh268 pms269 ? pmh269 pms270? pmh270 pms271 0-0.1 pmh271 pms272 ? pmh272 pms273 0-0.1 pmh273 pms2743 pmh274 pms275 ? pmh275 pms276 ? pmh276 pms277 4-5 pmh277 pms278 0-0.1pmh278 pms279 0-0.1 pmh279 pms280 0-0.1 pmh280 pms281 ? pmh281 pms282 ?pmh282 pms283 0-0.1 pmh283 pms284 ? pmh284 pms285 0-0.1 pmh285 pms2860.2 pmh286 pms287 0.2 pmh287 pms288 7 pmh288 pms289 4 pmh289 pms2900-0.1 pmh290 pms291 3.5 pmh291 pms292 1.5 pmh292 pms293 3 pmh293 pms2940-0.1 pmh294 pms295 1.5 pmh295 pms296 6 pmh296 pms297 4-5 pmh297 pms298? pmh298 pms299 ? pmh299 pms300 0-0.1 pmh300 pms301 0.2 pmh301 pms3020.3 pmh302 pms303 0.3 pmh303 pms304 0-0.1 pmh304 pms305 0-0.1 pmh305pms306 0.3 pmh306 pms307 ? pmh307 pms308 ? pmh308 pms309 0.5 pmh309pms310 0-0.1 pmh310 pms311 0.5 pmh311 pms312 6 pmh312 pms313 3 pmh313pms314 ? pmh314 pms315 ? pmh315 pms316 0-0.1 pmh316 pms317 ? pmh317pms318 ? pmh318 pms319 0-0.1 pmh319 pms320 ? pmh320 pms321 4-5 pmh321pms322 0.5 pmh322 pms323 ? pmh323 pms324 4 pmh324 pms325 0.2 pmh325pms326 2 pmh326 pms327 1 pmh327 pms328 0-0.1 pmh328 pms329 ? pmh329pms330 ? pmh330 pms331 4-5 pmh331 pms332 0.2 pmh332 pms333 ? pmh333pms334 2 pmh334 pms335 0-0.1 pmh335 pms336 0.2 pmh336 pms337 0-0.1pmh337 pms338 ? pmh338 pms339 0-0.1 pmh339 pms340 ? pmh340 pms341 0-0.1pmh341 pms342 ? pmh342 pms343 0-0.1 pmh343 pms344 ? pmh344 pms345 2pmh345 pms346 0-0.1 pmh346 pms347 0-0.1 pmh347 pms348 0.2 pmh348 pms3490.5 pmh349 pms350 0-0.1 pmh350 pms351 4 pmh351 pms352 0-0.1 pmh352pms353 0.3 pmh353 pms354 0-0.1 pmh354 pms355 0-0.1 pmh355 pms356 0-0.1pmh356 pms357 0-0.1 pmh357 pms358 0-0.1 pmh358 pms359 0-0.1 pmh359pms360 ? pmh360 pms361 0-0.1 pms361 pms362 ? pms362 pms363 ? pms363pms364 ? pms364 pms365 ? pmh365 pms366 0-0.1 pmh366 pms367 ? pmh367pms368 ? pmh368 pms369 0-0.1 pmh369 pms370 ? pmh370 pms371 0.5 pmh371pms372 0.3 pmh372 pms373 0.5 pmh373 pms374 ? pmh374 pms375 ? pmh375pms376 3 pmh376 pms377 ? pmh377 pms378 6 pmh378 pms379 ? pmh379 pms380 ?pmh380 pms381 4 pmh381 pms382 ? pmh382 pms383 ? pmh383 pms384 ? pmh384pms385 0-0.1 pmh385 pms386 ? pmh386 pms387 ? pmh387 pms388 ? pmh388pms389 ? pmh389 pms390 ? pmh390 pms391 0-0.1 pmh391 pms392 ? pmh392pms393 ? pmh393 pms394 ? pmh394 pms395 0-0.1 pmh395 pms396 0-0.1 pmh396pms397 0-0.1 pmh397 pms398 ? pmh398 pms399 ? pmh399 pms400 0-0.1 pmh400pms401 ? pmh401 pms402 0.2 pmh402 pms403 0-0.1 pmh403 pms404 0-0.1pmh404 pms405 0-0.1 pmh405 pms406 0-0.1 pmh406 pms407 ? pmh407 pms4080.5 pmh408 pms409 ? pmh409 pms410 0.5 pmh410 pms411 0-0.1 pmh411 pms4121 pmh412 pms413 0.2 pmh413 pms414 4.5 pmh414 pms415 6 pmh415 pms4160-0.1 pmh416 pms417 0-0.1 pmh417 pms418 4 pmh418 pms419 0-0.1 pmh419 ?pms420 2 pmh420 1.7 pms421 0.2 pmh421 6 pms422 0.5 pmh422 9 pms423 0-0.1pmh423 ? pms424 0-0.1 pmh424 0-0.1 pms425 1.2 pmh425 0-0.1 pms426 ?pmh426 0-0.1 pms427 4.5 pmh427 4 pms428 0-0.1 pmh428 7 pms429 6 pmh429 ?pms430 0.3 pmh430 3.5 pms431 0.5 pmh431 8 pms432 0-0.1 pmh432 1.5 pms4330.2 pmh433 4 pms434 0-0.1 pmh434 ? pms435 0-0.1 pmh435 7 pms436 0.2pmh436 4.4 pms437 0-0.1 pmh437 0-0.1 pms438 0-0.1 pmh438 6 pms439 0.5pmh439 5 pms440 0-0.1 pmh440 ? pms441 2 pmh441 7 pms442 ? pmh442 ?pms443 2.5 pmh443 ? pms444 4.5 pmh444 0-0.1 pms445 0.2 pmh445 2 pms4464.5 pmh446 ? pms447 3 pmh447 9-23 pms448 0-0.1 pmh448 1.5 pms449 0-0.1pmh449 3.2 pms450 0-0.1 pmh450 0-0.1 pms451 0-0.1 pmh451 0-0.1 pms4520.5 pmh452 ? pms453 0-0.1 pmh453 4.4 pms454 0.3 pmh454 1 pms455 0-0.1pmh455 1 pms456 0-0.1 pmh456 0-0.1 pms457 0-0.1 pmh457 ? pms458 3 pmh458? pms459 0.5 pmh459 ? pms460 4-5 pmh460 ? pms461 0-0.1 pmh461 ? pms4623-4 pmh462 ? pms463 0-0.1 pmh463 ? pms464 0-0.1 pmh464 ? pms465 0-0.1pmh465 ? pms466 5 pmh466 2.6 pms467 0-0.1 pmh467 ? pms468 6 pmh468 4.2pms469 0-0.1 pmh469 2.4 pms470 5 pmh470 4 pms471 9 pmh471 4.4 pms4720-0.1 pmh472 8 pms473 9-23 pmh473 3 pms474 0-0.1 pmh474 4 pms475 0.5pmh475 4.7 pms476 0-0.1 pmh476 ? pms477 5 pmh477 ? pms478 0-0.1 pmh478 ?pms479 0-0.1 pmh479 ? pms480 0.3 pmh480 2 pms481 1 pmh481 ? pms482 5pmh482 0-0.1 pms483 1.5 pmh483 ? pms484 9 pmh484 2.2 pms485 0.2 pmh4851.5 pms486 0-0.1 pmh486 2.2 pms487 0-0.1 pmh487 ? pms488 1.5 pmh488 2.2pms489 5 pmh489 ? pms490 0-0.1 pmh490 4 pms491 1.5 pmh491 1.5 pms4920-0.1 pmh492 4.8 pms493 0-0.1 pmh493 ? pms494 9 pmh494 ? pms495 0.2pmh495 ? pms496 0-0.1 pmh496 0-0.1 pms497 1.5 pmh497 0.5 pms498 0-0.1pmh498 ? pms499 0.3 pmh499 7 pms500 0.5 pmh500 0-0.1 pms501 0-0.1 pmh501? pms502 0-0.1 pmh502 2.6 pms503 0-0.1 pmh503 1.8 pms504 0-0.1 pmh5040.5 pms505 0-0.1 pmh505 2.2 pms506 3 pmh506 ? pms507 0.3 pmh507 1.8pms508 0.2 pmh508 ? pms509 3 pmh509 0.7 pms510 5 pmh510 2.3 pms511 0-0.1pmh511 ? pms512 0.5 pmh512 9-23 pms513 0-0.1 pmh513 1.5 pms514 4 pmh514? pms515 0-0.1 pmh515 3.2 pms516 6 pmh516 8 pms517 0-0.1 pmh517 ? pms5180-0.1 pmh518 0-0.1 pms519 0.2 pmh519 3.5 pms520 0-0.1 pmh520 4.4 pms5210.3 pmh521 ? pms522 5 pmh522 5 pms523 0.6 pmh523 4.4 pms524 0.3 pmh524 7pms525 0-0.1 pmh525 1.5 pms526 6 pmh526 5.5 pms527 0-0.1 pmh527 1.5pms528 6 pmh528 7 pms529 0.2 pmh529 4.4 pms530 3 pmh530 7 pms531 3pmhs31 2.2 pms532 0-0.1 pmh532 8 pms533 0-0.1 pmh533 9 pms534 5 pmh5341.5 pms535 0-0.1 pmh535 ? pms536 5 pmh536 1.5 pms537 0-0.1 pmh537 ?pms538 2 pmh538 ? pms539 0-0.1 pmh539 2.2 pms540 0-0.1 pmh540 ? pms5410-0.1 pmh541 ? pms542 0-0.1 pmh542 4.6 pms543 3 pmh543 0-0.1 pms544 4pmh544 1.5 pms545 0-0.1 pmh545 8 pms546 0-0.1 pmh546 9 pms547 0-0.1pmh547 1.5 pms548 1.5 pmh548 0.2 pms549 3 pmh549 0.5 pms550 4.4 pmh550 ?pms551 0-0.1 pmh551 0-0.1 pms552 0-0.1 pmh552 5.5 pms553 0-0.1 pmh553 3pms554 0-0.1 pmh554 0-0.1 pms555 4.4 pmh555 7 pms556 0-0.1 pmh556 ?pms557 0-0.1 pmh557 ? pms558 3 pmh558 2.6 pms559 0-0.1 pmh559 ? pms5600.5 pmh560 ? pms561 0-0.1 pmh561 0-0.1 pms562 2 pmh562 4 pms563 0-0.1pmh563 2.2 pms564 0-0.1 pmh564 ? pms565 0-0.1 pmh565 ? pms566 6 pmh566 ?pms567 9 pmh567 ? pms568 0.3 pmh568 ? pms569 0.5 pmh569 ? pms570 0.2pmh570 5 pms571 0.5 pmh571 4 pms572 0-0.1 pmh572 ? pms573 0-0.1 pmh573 ?pms574 0.2 pmh574 7 pms575 0.3 pmh575 5.5 pms576 3 pmh576 8 pms577 0.5pmh577 9 pms578 0-0.1 pmh578 0-0.1 pms579 0-0.1 pmh579 9 pms580 0-0.1pmh580 0-0.1 pms581 ? pmh581 ? pms582 ? pmh582 0-0.1 pms583 ? pmh583 5pms584 ? pmh584 4 pms585 ? pms586 ? pms587 ? pms588 ? pms589 ? pms5900-0.1 pms591 0-0.1 pms592 ?

14 1461 base pairs nucleic acid single linear unknown 1 GTCGACAGACTACTAACTTC AGCCTATCTA GTAAAACAAG CTAAAAGATT CGACGGAGTT 60 GACCCAGCCTTCCCTGCCGC CCTCACCTGC GCTTCTCACC TCATGCTTTC TTCCATGGAT 120 TCCCATACAAAGTCATCTTT CATGGACAAC ATCAAATTGC ACATGACTGA TACTCAATGC 180 TTCTTCAAGAACATTGAACG ATTTGAGAAA TTCTTGGGAA GATATGGGGA CGAATACGCC 240 ATGTCCCACAAGCAAAATTG TAACTGCCCC TTCCATCTCC ACCACACTTT TACTCCCTCA 300 GATAACGAGCATCTGGTATC CTCTTTCGCA TTCGCCCGCC CAGAAGTCTC CATGGAAGAA 360 ATTAGAGCCACACCCTATCA GGCCAACAAG CTTATTAGTG ACAAACATTA CGTGATGAAC 420 ATGTCCAAGATCGATTCTAG AGTAACAGGA TCTTCCCTCC TTAAGAAGGT TAGCGAATGG 480 ACTGAAATGAGAATGAACTC CAACTTTAAT GGAACATTTG AACCATCAAG ACTCGCCCTC 540 TCCAACTCTGGCATGACAAC GGCAGGAGTC AACCTCGACG TTATTGTCAA ACCAAATAAT 600 GCAAGAAGTGTACTAGGAAT ATTGGAATGT CATCGCCAGC ACGTGTGCAC CGCCGACGCC 660 AAGGGAACTGTCGCTTCAGC CATGCCAGCC GTCTTCCAGG CAACCGATGG AAACGGTAAC 720 GAATCTGAACTGATCCAGAA TGCTCTGCCA AGGAACAGAT ACATCCAAAA GAGCACAATG 780 AACGCTCAAACTGTCGTGTT TGCTAATGTT TTGGAACAAC TTATCGCCGA TCTTGGAAAG 840 GTTATCGTGAACGAACTGGC CGGCACCATC GCTGAATCTG TACCAGAAAG CGTATATGAA 900 AACACCAAGGAAATGATTGA TAGACTAGGC TCTGACGACC TCTTCAAATC TAATAATAAT 960 GGAGGAGTAGAATCAATGGA TTATGAAGAT AGCGAAACAA CATCCAACAA TGGTCCCGTC 1020 CTCATCTCAGAAGCCATGAA GAATGCCGTC TATCACACAC TAATTTCCGG CAAGGCAGCT 1080 CGCCCGGAAAATGTACCATT CGCCTCATGC GCCAGCGGCC CTCTCGCCTT TGATTTCCTT 1140 CTGTCAAAGGGAGATACATT CGAAGAAAAG AACGCCGAAC AAGGTGCAGC AGCTGCCGTA 1200 TCCTCTACCTATTCTTCCTC TTCTAACACT ACTCTTCGTA AGCATTTGGC TCGAGTTTTC 1260 GAAGCCATCTCTAAGCAAGT AACTGATGCT GAATTCAAGG ATATCCTCAA CGATATCGAA 1320 CGTAATATTTCTTCTGACTA TACTAACTGT CCACCAAATA CTAACCAAAA TGCCTTTGCT 1380 CTAGCTATCAAGAGAGAATT CAGCAGAATT GTTTCCTTCT TAACCATTCT TCGTAAGAAC 1440 ATTACACCCGCATTAGTCGA C 1461 23 base pairs nucleic acid single linear unknown 2ACTACTAACT TCAGCCTATC TAG 23 25 base pairs nucleic acid single linearunknown 3 TAATGCGGGT GTAATGTTCT TACGA 25 22 base pairs nucleic acidsingle linear unknown 4 GTAACTGCCC CTTCCATCTC CA 22 22 base pairsnucleic acid single linear unknown 5 TACGGCAGCT GCTGCACCTT GT 22 21 basepairs nucleic acid single linear unknown 6 TGGGAAGATA TGGGGACGAA T 21 23base pairs nucleic acid single linear unknown 7 CGAAGAGTAG TGTTAGAAGAGGA 23 21 base pairs nucleic acid single linear unknown 8 AGAAGGTTAGCGAATGGACT G 21 21 base pairs nucleic acid single linear unknown 9TTGAAGAGGT CGTCAGAGCC T 21 23 base pairs nucleic acid single linearunknown 10 GAAACGGTAA CGAATCTGAA CTG 23 18 base pairs nucleic acidsingle linear unknown 11 CAGTCCATTC GCTAACCT 18 18 base pairs nucleicacid single linear unknown 12 CGTCCCCATA TCTTCCCA 18 24 base pairsnucleic acid single linear unknown 13 TGCCTTATCA GCTNTCGATT GTAG 24 24base pairs nucleic acid single linear unknown 14 TTCAGNTTTG CAACCATACTTCCC 24

What is claimed is:
 1. An isolated non-occluded baculovirus associatedwith white spot syndrome in an arthropod host, the baculovirus beingidentical to that deposited in the China Center for Type CultureCollection as Accession Number CCTCC-V96001.
 2. An isolated non-occludedbaculovirus associated with white spot syndrome in an arthropod host,the baculovirus comprising a genomic DNA molecule comprising thenucleotide sequence of SEQ ID NO:1.
 3. An isolated nucleic acidcomprising the nucleotide sequence of SEQ ID NO:1 or the complementthereof.
 4. A nucleic acid segment of a nucleic acid consisting of thesequence of SEQ ID NO:1 or the complement thereof, wherein the segmentis 18-25 base pairs in length.
 5. The nucleic acid segment of claim 4,further comprising a digoxigenin molecule.
 6. The nucleic acid segmentof claim 4, wherein the segment consists of a nucleic acid sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, and SEQ ID NO:6.
 7. A method of detecting a baculovirus infectionin a shrimp, the method comprising a) providing a sample obtained from ashrimp; and b) detecting the presence of a baculovirus in the sample byi) Southern hybridization using the nucleic acid segment of claim 4, ii)dot blotting using the nucleic acid segment of claim 4, or iii)performing a polymerase chain reaction using at least one nucleic acidsegment of claim 4 as a primer.
 8. The method of claim 7, wherein thenucleic acid segment used for dot blotting is labeled with digoxigenin.9. An isolated nucleic acid comprising the nucleotide sequence of SEQ IDNO:2 or the complement thereof.
 10. A nucleic acid segment of a nucleicacid consisting of the sequence of nucleotides 9 to 1455 of SEQ ID NO:1,or the complement thereof, wherein the segment is 18-25 base pairs inlength.
 11. The nucleic acid segment of claim 10 further comprising adigoxigenin molecule.
 12. A method of detecting a baculovirus infectionin a shrimp, the method comprising a) providing a sample obtained from ashrimp; and b) detecting the presence of a baculovirus in the sample byi) Southern hybridization using the nucleic acid segment of claim 10,ii) dot blotting using the nucleic acid segment of claim 10, or iii)performing a polymerase chain reaction using at least one nucleic acidsegment of claim 10 as a primer.
 13. The method of claim 12, wherein thenucleic acid segment used for dot blotting is labeled with digoxigenin.